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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Invest Ophthalmol Vis Sci. Author manuscript; available in PMC 2009 October 1.
Published in final edited form as:
PMCID: PMC2720629
NIHMSID: NIHMS118735

Relative course of retinal nerve fiber layer (RNFL) birefringence, RNFL thickness and retinal function changes after optic nerve transection

Abstract

Purpose

To test the hypothesis that alterations of RNFL birefringence precede changes in RNFL thickness (RNFLT) in an experimental model of RGC injury. Secondarily, to determine the time course of RGC functional abnormalities relative to RNFL birefringence and RNFL thickness changes.

Methods

RNFL birefringence was measured by scanning laser polarimetery (GDx VCC, Carl Zeiss Meditec, Inc). RNFL thickness was measured by spectral domain optical coherence tomography (sd-OCT, Spectralis™ HRA+OCT, Heidelberg Engineering, GmbH). Retinal function was assessed by three forms of electroretinography (ERG): slow-sequence multifocal ERG (mfERG, VERIS, EDI); pattern-reversal ERG (PERG, Utas-E3000, LKC Technologies, Inc); and photopic full-field flash ERG (ff-ERG, Utas-E3000). All measurements were obtained in both eyes of four adult rhesus macaque monkeys (Macaca mulatta) during two baseline sessions, and again 1-week and 2-weeks after unilateral optic nerve transection (ONT).

Results

ONT was successfully completed in 3 subjects. RNFL birefringence declined by 15% one week after ONT (p = 0.043), while there was no significant change in RNFL thickness (+1%, p = 0.42). Two weeks after ONT, RNFL retardance had declined by 39% (p = 0.018) while RNFL thickness had declined by only 15% (p = 0.025). RGC functional abnormalities were present 1-week after ONT, including decreased amplitudes relative to baseline of the mfERG high frequency components (−65%, p = 0.018); the PERG N95 component (−70%, p = 0.007) and the photopic negative response of the ff-ERG (−44%, p = 0.005).

Conclusions

RNFL birefringence declined prior to, and faster than RNFL thickness after ONT. RGC functional abnormalities were present 1-week after ONT, when RNFL thickness had not yet begun to change. RNFL birefringence changes after acute RGC injury are associated with RGC dysfunction. Together, they reflect RGC abnormalities that precede axonal caliber changes and loss.

Keywords: retinal ganglion cell, retinal nerve fiber layer, scanning laser polarimetry, birefringence, optical coherence tomography, electroretinogram

Introduction

Previous studies have shown that cytoskeletal components and their tertiary structure within retinal ganglion cell (RGC) axons cause the retinal nerve fiber layer (RNFL) to exhibit the optical property of form birefringence.13 This is supported both by theoretical analyses1,2 and by evidence demonstrating that RNFL birefringence rapidly declines after chemical disruption of cytoskeletal components, microtubules (MT) in particular, in situ3 or in vivo.4 This has clinical relevance because cytoskeletal abnormalities might develop in diseases such as glaucoma prior to RGC death. For example, in experimental models of RGC injury such as optic nerve transection (ONT) or crush, there is a delay among the majority of surviving RGCs before axonal caliber begins to decline,5 which is preceded by changes in cytoskeletal protein content and mRNA.6,7 Abnormalities of cytoskeletal proteins such as neurofilament (NF) have also been demonstrated in experimental models of glaucoma 810 and may represent a mechanism of susceptibility.11 Thus, it is possible that measurement of RNFL birefringence could be used to detect early stage cellular dysfunction and/or to predict subsequent risk of progression and permanent loss.3,12

RNFL birefringence can be measured clinically using scanning laser polarimetry (SLP)1214 or polarization sensitive optical coherence tomography.1518 Using SLP, Mohammadi et al19 found that measures of RNFL birefringence were an independent predictor of future vision loss in glaucoma suspects who began the study with normal SAP visual fields, regardless of their age, IOP, or optic disc appearance. Although this is consistent with the hypothesis that cytoskeletal abnormalities were present prior to subsequent progressive changes in optic nerve structure or function, the technique of SLP measures retardance, which is a function of both RNFL birefringence and RNFL thickness.3,12,13,17,18,20,21 Thus it is possible that in the Mohammadi et al study19 RNFL thickness changes were present at baseline and predictive of future glaucomatous progression.22

The primary purpose of the present study was to test the hypothesis that alterations of RNFL birefringence precede changes in RNFL thickness in an experimental model of RGC injury. The secondary purpose was to determine the time course of RGC functional abnormalities relative to RNFL birefringence and RNFL thickness changes.

Methods

Subjects

The subjects of this study were four adult rhesus macaque monkeys (Macaca mulatta). Table 1 lists the age, weight and sex of each animal. All experimental methods and animal care procedures adhered to the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local Institutional Animal Care and Use Committee (IACUC).

Table 1
Study subjects age, weight, sex and intraocular pressure.

Anesthesia

All experimental procedures began with induction of general anesthesia using ketamine (15 mg/kg IM), along with a single subcutaneous injection of atropine sulphate (0.05 mg/kg). Animals were then intubated and breathed 100% oxygen for retinal function testing by electroretinography (ERG), during which anesthesia was maintained using a combination of ketamine (5 mg/kg/hr IV) and xylazine (0.8 mg/kg/hr IM). Upon completion of retinal function testing, ketamine-xylazine administration was discontinued and isoflurane gas (1–3%) was mixed with oxygen to provide anesthesia during structural imaging of the retina and optic nerve head (ONH). Isoflurane (1–1.5%) was also used to provide anesthesia during optic nerve transection surgery.

During all procedures, heart rate and arterial oxyhemoglobin saturation were monitored continuously (Propaq Encore model 206EL, Protocol Systems, Inc., Beaverton, OR) and maintained above 75 min−1 and 95%, respectively. Body temperature was maintained with a warm-water heating pad set at 37°C.

Retinal Function Testing

Retinal function was evaluated by three different modes of ERG as previously described.23,24 Custom-designed Burian-Allen contact lens electrodes (10 mm diameter, +3.0 diopter; Hansen Ophthalmics, Iowa City, IA) were used for all ERG testing; the corneal ring on the stimulated eye served as the active electrode, while the corneal ring of the unstimulated (patched) contralateral eye served as the reference electrode. A subcutaneous ground electrode was placed on a rear limb. Electrode impedance was accepted if <5 kΩ. Prior to insertion of ERG contact lens electrodes, one drop of topical anesthetic (0.5% proparacaine) and an ocular lubricating agent (Celluvisc; Allergan, Irvine, CA) were applied to each eye. Head position was stabilized using a bite bar apparatus capable of rotation in three axes.

Multifocal ERGs were recorded using VERIS™ (version 4; EDI, San Mateo, CA). Residual refractive error was measured by retinoscopy for the test distance (25 cm) and corrected to the nearest half diopter. The mfERG stimulus was presented on a 21-inch monochrome monitor with a 75 Hz refresh rate. An initial set of brief recordings (2 min each) was used to center the stimulus on the visual axis such that the foveal and “blind spot” responses were positioned appropriately within the response array.

The mfERG stimulus consisted of 103 un-scaled hexagonal elements subtending a total field size of ~55 degrees. The luminance of each hexagon was independently modulated between dark (1 cd/m2) and light (200 cd/m2) according to a pseudorandom, binary m-sequence. Stimulus luminance was measured using a calibrated spot photometer (SpectraScan PR-650, Photo Research). The temporal stimulation rate was slowed by insertion of 7 dark frames into each m-sequence step (“7F”). The m-sequence exponent was set to 12, thus the total duration of each recording was 7 min 17 sec. Signals were amplified (gain = 100,000), band-pass filtered (10–300 Hz; with an additional 60 Hz line filter), sampled at 1.2 kHz (i.e. sampling interval = 0.83 ms), and digitally stored for subsequent off-line analyses. Two such recordings were obtained for each eye at each time point and averaged.

From the average of the two recordings at each time point, a subset of local responses was exported for further analyses. Figure 1A shows the stimulus locations of this subset, consisting of the central part of the array where RGC contributions are largest.24,25 The response from the central stimulus element (marked with a “C”) and those from the two surrounding concentric rings were evaluated: locations are numbered 1–6 around the first ring; and 1–12 around the second ring.

Figure 1
Multifocal ERG stimulus pattern (A). Gray-colored oval indicated position of the “blind spot” within the stimulus field for left eye recordings. Numbered locations show position of responses that were exported for analysis: “C”, ...

Each local mfERG response was band-passed filtered (−3 dB at 65 and 250 Hz) to extract the high frequency components (HFC, Fig. 1B). The low frequency component (LFC) of each response was represented as the raw response minus the HFC. The amplitude of the HFC was calculated as the root mean square (RMS) for the epoch between 0–80 ms of each filtered record. Peak amplitudes for LFC features (see Fig. 1B) were quantified as follows: the first negative feature (N1) was calculated as the maximum negative excursion from baseline in the epoch up to 30 ms; the amplitude of the first positivity (P1) was calculated as the voltage difference between the maximum peak and the N1 trough; and the second negativity (N2) was calculated as the difference between baseline and the minima from 30–80 ms.

Pattern-reversal ERGs

Transient pattern-reversal ERGs (PERGs) were recorded using a Utas-E3000 system (LKC Technologies, Inc, Gaithersburg, MD). The PERG stimulus was a checkerboard pattern (1 degree check size), reversing at 2.5 Hz (5 reversals/sec). The stimulus subtended 32 × 24 deg at the 50 cm test distance. Stimulus luminance was 75 cd/m2 and contrast was >90%. The position of the foveal projection determined during mfERG testing (see above) was used to align the center of the PERG stimulus on the visual axis. Residual refractive error was measured for the test distance and corrected to the nearest half diopter. Signals were band-pass filtered 1–500 Hz and sampled at 2 kHz. Two records were obtained for each eye and then averaged. Each single record was an average of 200 sweeps. Eye position was monitored continuously and remained stable with sufficient depth of anesthesia. Amplitudes were measured for the primary features commonly known as P50 and N95 (see Fig. 2A); the P50 was calculated as the difference between the peak and baseline, the N95 was calculated as the difference between the peak around 50 ms and the trough (minimum) around 95 ms.

Figure 2
Representative individual example (ONT3) of PERG (A) and photopic full-field flash ERG responses (B). Left eye (control) responses are shown as broken traces while responses from the right (experimental) eye are shown as solid traces. Responses are repeatable ...

Full-field flash ERGs

Using the same UTAS-E3000 system, photopic full-field flash ERGs (ffERGs) were obtained after 5 min of light adaptation to a rod-saturating blue background (30 scotopic cd/m2; Wratten #78; Eastman Kodak Co., Rochester, NY). Red stimulus flashes (Wratten #29) with an intensity of 0.42 log photopic cd.s/m2 were presented monocularly at 0.5 Hz via a ganzfeld integrating sphere. Stimulus and background intensities were measured using a calibrated photometer (Spectra Pritchard PR-1980A, Photo Research, Chatsworth, CA). Signals were band-pass filtered 0.3–500 Hz and sampled at 2 kHz. Two records were obtained and then averaged. Each single record was an average of 10 sweeps.

Amplitudes were measured for four features, the a-wave, b-wave, oscillatory potentials (OPs) and photopic negative response (PhNR; see Fig. 2B). The a-wave amplitude was measured at the criterion time of 10 ms after the stimulus flash; the b-wave as the difference between the peak and the a-wave trough values (i.e. a peak-to-trough amplitude); the PhNR as the difference between the value at the criterion time of 85 ms after the stimulus flash and the value at the b-wave peak. OPs were isolated using a Blackman filter (−3 dB at 65 and 240 Hz) and their summed amplitude was quantified as the RMS of the filtered waveform between zero and 100 ms.

Clinical imaging of retinal and ONH structure

RNFL birefringence. Retardance measurements were obtained by scanning laser polarimetry (SLP) using a GDxVCC instrument (Carl Zeiss Meditec, Inc.). The instrument compensates for the effects of anterior segment (primarily corneal) retardance to more accurately determine RNFL retardance.14,26 Thus, anterior segment retardance measurements are obtained prior to initial baseline RNFL scans, then used to compensate all subsequent RNFL scans. A bite-bar, which rotates in three axes, was used to properly align the head and eye, and auto-refraction is used for each scan. Three RNFL scans were averaged for each eye at each time point.

Figure 3 provides an individual example (ONT3) of RNFL retardance data obtained by SLP. The pseudo-color maps in 3A–3D represent retardance as RNFL “thickness” in microns. The GDxVCC instrument detects the retardance of a cross-polarized source after a double pass through the tissue sample, assumes that RNFL thickness is linearly related to retardance, then calculates and reports an estimate of RNFL “thickness” using a linear conversion factor of 0.67 nm/µm13 (as stated in the instrument manual27).

Figure 3
RNFL birefringence maps for one representative eye obtained by SLP during two baseline sessions (A and B, respectively), one week after ONT (C) and two weeks after ONT (D). TSNIT curves for RNFL birefringence in the same eye at the same four time points ...

Values of RNFL birefringence were exported for the “small” peripapillary locus (the SLP instrument’s default). The exported data consist of 64 samples along a peripapillary locus beginning on the temporal side of the ONH, proceeding around the superior, nasal, inferior aspects of the ONH and completing a circle at the temporal location, thus representing a profile commonly referred to as a “TSNIT” curve (e.g. Fig. 3E). Each of the values in the TSNIT curve is an average from an 8-pixel wide band centered on the optic disc.27 This band is indicated by the pair of gray circles concentric around the ONH on each of the pseudo-color birefringence maps in Fig. 3A–3D. The inner and outer limits of the band are 27 and 35 pixels from the center of the optic disc, so the center of the band has a radius of 31 pixels.27 This corresponds to a scan angle with a radius of 6.1 degrees,27 which translates to about 1.12 mm on the macaque retina (assuming an emmetropic eye with average axial length of 19 mm).28,29

RNFL thickness measurements were obtained by spectral-domain optical coherence tomography (sd-OCT) using a Spectralis™ HRA+OCT instrument (Heidelberg Engineering, GmbH, Heidelberg, Germany). The optical resolution of the instrument is ~7 µm axially (depth) and ~14 µm transversely. The depth of each a-scan is 1.8 mm and consists of 512 pixels providing a digital depth sampling of 3.5 µm per pixel. Each b-scan spans 15 deg and consists of 768 a-scans providing a digital transverse sampling of 5 µm per pixel (in an emmetropic human eye with average axial length). For this experiment, volume scans consisting of 145 horizontal b-scan sections were centered on the ONH. Each b-scan in the volume spanned 15 deg horizontally and the block of 145 b-scans spanned 15 deg vertically (thus b-scans were separated by 0.1034 deg vertically). Radially oriented b-scans were also acquired with 48 sections arranged in a “star” pattern centered on the ONH.

A real-time eye tracking system measures eye movements and provides feedback to the sd-OCT scanning system in order to stabilize the retinal position of the b-scan. This system thusly enables sweep averaging at each b-scan location to reduce speckle noise. For this experiment, nine sweeps were averaged for each b-scan.

RNFL thickness measurements were derived from manual delineation of anterior (internal limiting membrane) and posterior borders along a single a-scan at the appropriate eccentricity within each radial b-scan. This eccentricity was chosen to correspond with the location of the RNFL birefringence measurement acquired by SLP. This eccentricity was determined to be equivalent to “1400 µm” from the center of the ONH as indicated by a ruler within the OCT visualization software. In converting angular span to linear distance, the Spectralis instrument assumes an emmetropic human eye with average axial length. This dimension translates to ~1120 µm on the macaque retina (assuming 19 mm axial length)28,29 and thus corresponds to the locus of SLP birefringence measurements. Figure 4 provides an example of the RNFL thickness measurement method. The radial b-scans were used for all RNFL thickness measurements reported here. Cross-validation was performed using the horizontal volume scans where measurements at the same eccentricity and polar angle differed by ≤5 µm (~1 pixel) from those made within radial b-scans.

Figure 4
Representative example demonstrating method of RNFL thickness measurement by sd-OCT. Measurements were obtained at two locations along each of 80 radial sections centered on the ONH. The position and direction of the vertical section is indicated by the ...

Confocal scanning laser tomography (CSLT) was perfomed using a Heidelberg Retina Tomograph (HRT II). During each imaging session, seven 15×15 deg scans were acquired, from which at least three were used to create a mean.3035 The effect of ONT on ONH surface topography was evaluated by two methods of analysis: Topographic Change Analysis (TCA)36,37 and the parameter Mean Position of the Disc (MPD).32,33

Simultaneous-stereoscopic photographs (3-Dx, NIDEK Co., Ltd., Aichi, Japan) of the ONH and peripapillary retina were obtained at baseline and at the final time point (2 weeks post ONT).

Digital video fluorescein angiography was performed one week after ONT using the Spectralis™ HRA+OCT instrument to evaluate retinal and choroidal circulation. After brief recording of background fluorescence, 0.8 ml of 10% sodium fluorescein (100 mg/ml, 80 mg total dose, Fluorescite, Alcon Laboratories, Inc. Fort Worth Texas) was injected intravenously as the clock timer was set to zero.

Intraocular pressure (IOP) measurements were made at the start of each session using a Tonopen™ (Oculab, Inc., Glendale, CA). The value for each eye was taken as the average of three successive measurements.

Optic nerve transection surgery was performed under general anesthesia (isoflurane, as described above). Complete transection of the intraorbital, retrobulbar optic nerve was achieved under direct visualization via lateral orbitotomy. Tissues were closed on multiple planes with 3−0 Nylon sutures, intraorbital and subcutaneous antibiotics were administered upon closure. Pain medications were administered for the first five post-operative days in conjunction with veterinary staff.

Experimental design and protocol

Two baseline sessions of retinal and ONH structural imaging (SLP, OCT, CSLT) and two baseline measurements of retinal function testing by ERG were completed in each eye prior to ONT surgery. ONT surgery was then performed on the right eye of each of the four animals in this study. Structural imaging and ERG testing was performed one week (7 days) and two weeks (14 days) after ONT. Animals were sacrificed 14 days after ONT by barbituate overdose and ocular tissues were harvested for a separate, ongoing study on proteomics. Thus, retinal and optic nerve tissues were not available for histopathological evaluation for this study.

Statistics

Repeated-Measures Analysis of Variance (RM-ANOVA) was used to test for effects of treatment (ONT) and time (Prism v4, GraphPad Software, Inc). Posthoc tests of differences between time points were performed as paired t-tests with Bonferroni’s correction for multiple comparisons.

Results

ONT surgery was successfully accomplished in three of the four animals. In the fourth (ONT4, Table 1) an intraoperative hemorrhage was observed within the orbit at the moment of transection, suggesting involvement of the central retinal artery (CRA) despite the transection being ~7 mm posterior to the globe. Fluorescein angiography 7 days after surgery confirmed that there was no direct perfusion of the CRA, therefore this animal was excluded from the study. Figure 5 shows a single frame from the angiograph obtained one week after ONT in each of the four experimental eyes. Panels 5A–5C each show a frame taken during venous laminar filling phase, which demonstrate normal circulation for the three subjects included, while panel 5D shows the final frame of the angiograph for the excluded subject. Table 1 lists the IOP for each eye at each time point. ONT had no effect on IOP (p = 0.72, RM-ANOVA) and there was no IOP difference between right eyes (ONT) and left eyes (control) at any time point (p > 0.05 for all post hoc tests).

Figure 5
Single frame taken from a digital video fundus fluoroscein angiograph one week after ONT in each of the four subjects (ONT1, A; ONT2, B; ONT3, C; ONT4, D). Frame taken from laminar venous filling phase of angiograph in A–C and from the final frame ...

Figure 6 shows the TCA Change Probability Maps derived from CSLT data for subject ONT3, which were used to evaluate changes in ONH surface topography after ONT. The first two panels (6A and 6B) show the reflectance images obtained during each of two baseline sessions. Figs. 6C and 6D show the Change Probability Maps overlaid onto the reflectance images obtained during the 1-week and 2-week post ONT sessions, respectively. Red and green colored super pixels indicate that a significant change in height has occurred (with a probability of p<0.05) at that location relative to baseline height and variability. Green colored super-pixels indicate elevation, and red pixels indicate depression relative to baseline height. Figure 6 demonstrates that there was little change in ONH surface topography after ONT. Across the group of three subjects, there was no significant effect of ONT on the MPD parameter (p = 0.84, RM-ANOVA). However, Fig. 6D does show that the brightness of the RNFL striations began to decrease, indicating a decrease in RNFL reflectance 2 weeks after ONT.

Figure 6
Representative example of CSLT TCA map (ONT3). First baseline session (A); second baseline session (B); one week after ONT (C); two weeks after ONT (D). Red pixels indicate significant depression relative to baseline height, green pixels indicate significant ...

Figure 3 provides an individual example (ONT3) of RNFL birefringence and RNFL thickness changes after ONT measured by SLP and sd-OCT, respectively. Figs. 3A and 3B show the birefringence maps obtained during the two baseline time points and Figs. 3C and 3D shows the maps obtained at one week and two weeks after ONT, respectively. The TSNIT curves derived from the SLP data for this eye at each time point are shown in Fig. 3E. The TSNIT average value at each time point was as follows: 53.7, 57.0, 48.7, and 28.0 µm. Thus, there was a 12% decline in RNFL retardance from the average baseline value one week after ONT and a 49% decline from baseline two weeks after ONT. Figure 3F shows the TSNIT curves for the fellow control eye obtained during the same four time points. The TSNIT average values were 62.1, 59.9, 53.3, and 61.5, representing a coefficient of variation (COV) in this control eye of 6.8%.

Figures 3G and 3H show the TSNIT curves for RNFL thickness measured by sd-OCT in the same subject (ONT3). The coefficient of variation across the four time points in the control eye was 0.9%, better than that for retardance. The TSNIT average values for RNFL thickness in the experimental eye were: 100.1, 112.8, 101.9, and 85.1 µm, indicating that RNFL thickness declined by 4% one week after ONT and by 20% two weeks after ONT. Fig. 3G also demonstrates emergence of the retinal blood vessels as RNFL thickness begins to decline and recede around the vessels, note how spikes in the TSNIT curve become prominent but maintain consistent position and thickness as compared with baseline.

Figure 7 shows the results for the group of three subjects. The average TNSIT values for each eye at each time point were normalized to the mean of the two baseline values. Figure 7A shows that RNFL retardance declined by 15% one week after ONT (p = 0.043), while there was no significant change in RNFL thickness (+1%, p = 0.42). Two weeks after ONT, RNFL retardance had declined by 39% (p = 0.018) while RNFL thickness had declined by only 15% (p = 0.025). Figure 7B shows that there were no significant changes in the group of fellow control eyes for either RNFL retardance (p = 0.16) or RNFL thickness (p = 0.97). The data in Fig. 7B also confirm that RNFL thickness measurements had better inter-session repeatability (average COV = 2.9%) than RNFL retardance measurements (average COV = 5.9%) among control eyes.

Figure 7
Group average (n=3) RNFL measurements for the 360 degree peripapillary scan circle (TSNIT curves) obtained during each of two baseline sessions (BL1 and BL2 respectively), one and two weeks after ONT (wk1 and wk2, respectively) in the experimental eyes ...

Figure 1 shows mfERG responses for the same individual subject ONT3. Fig. 3C and 3D show the mfERG responses obtained during baseline from the left eye (control) and right eye (experimental), respectively. The HFCs are prominent in responses from both eyes and a strong nasal-temporal asymmetry is evident as the stimulus location changes around the two rings surrounding the central location. Figure 3E shows the responses from the same locations one week after ONT was performed in the right eye, while 3F shows the responses from the fellow control eye that same day. The amplitude of the HFCs had declined substantially (66% on average across the 19 locations) one week after ONT. The response morphology had also become more similar around the rings, reflecting a decrease of the nasal-temporal asymmetry. The amplitude of LFC features also became smaller after ONT, though to a lesser extent than the HFC.

Figure 2 shows the PERG (panel A) and ff-ERG results (panel B) for the same individual subject ONT3. There is a marked decline in the amplitude of the PERG N95 component one week after ONT (91%, see arrows) as compared with either the baseline responses from the same eye or the responses from the fellow control eye (dashed curves). The effect of ONT on the PERG P50 component in this eye was to speed the apparent implicit time (by ~2–4 ms), but the P50 amplitude did not decrease as much as the N95.

Similarly, Figure 2B demonstrates that ONT had a strong and relatively selective effect on the amplitude of the ff-ERG PhNR (see arrows), as compared with either the baseline responses from the same eye or the responses from the fellow control eye (dashed curves). ONT had a larger effect on the PhNR as compared with the a-wave, b-wave or OP’s.

Table 2 lists the results of retinal function testing for the group of three subjects. The values listed for 1-week and 2-weeks post ONT show the change from the average baseline for each parameter, each value represents the mean change for the 3 ONT eyes. The results of statistical testing are also listed (two-way RM-ANOVA). The probabilities in the first column represent the chance that the “treatment” effect (ONT) was due to random variation rather than to ONT; the probabilities in the second column represent the chance that the observed interaction between time and treatment (ONT) is due to random variation. The last column lists the COV calculated for the group of 3 fellow control eyes for each parameter, which provides a basis for comparing the observed effect of ONT against normal intersession variability. The results in Table 2 demonstrate that there were significant changes in retinal function 1 week after ONT. The largest effects of ONT were on the mfERG HFC, the PERG N95 component and the PhNR of the photopic ff-ERG. There was a trend toward recovery of function 2 weeks after ONT for the N1 component of the mfERG N1 and the a-wave of the photopic ff-ERG.

Table 2
Results of retinal function testing. Values listed for 1-week and 2-weeks post ONT represent the change from the baseline average (mean of n=3 subjects). Probabilities listed in each row are derived from a two-way RM-ANOVA; the first column represents ...

Discussion

The results of this study demonstrate that RNFL birefringence decreased one week after ONT while RNFL thickness had not yet changed. By the second week after ONT, RNFL thickness had declined by 15% while the decrease in retardance measured by SLP was more than twice that amount (39%), suggesting that RNFL birefringence (retardance per unit thickness) had declined further still.3,12,13,17,18,20,21 It is thought that form birefringence of the RNFL is due to the orderly structural array of thin cylindrical cytoskeletal components within RGC axons such as MT and NF.13 Previous studies have shown that RNFL birefringence declines rapidly after chemical disruption of MT, a component of the RGC cytoskeleton, in situ3 or in vivo.4 Thus it has been suggested that measurements of RNFL birefringence could provide a sensitive indicator of compromised cytoskeleton within RGC axons.3,12 The results of this study indicate that there is a stage during RGC degeneration where the axonal cytoskeleton has become abnormal enough to result in altered RNFL birefringence, and that this stage precedes thinning of the axon bundles of the peripapillary RNFL.

This intermediate stage observed in this study in response to a relatively acute experimental injury (ONT), might be common to other forms of RGC injury, such as in glaucoma. For example, it has been suggested that reduced gene NF expression represents a general response to RGC injury.7 Moreover, abnormalities of cytoskeletal proteins such as NF and MT have been demonstrated in experimental models of glaucoma8,11 and after short-term elevation of IOP.9,10,38,39 This is important partly because critical functional capabilities depend on intact MT as they provide the “tracks” upon which most active axoplasmic transport takes place.4042 Interruption of axoplasmic transport has been proposed as a fundamental pathophysiological process in glaucoma and has been demonstrated to occur during acutely and chronically elevated IOP states in several mammalian species.9,10,38,39,4346 Chemical disruption of MT by colchicine or vinblastine halts axonal transport in RGCs.40,41 Therefore, MT disruption may not only be caused by elevated IOP, but also lead to further abnormalities of axoplasmic transport, perhaps resulting in a vicious cycle. Further studies are underway to determine whether a similar intermediate stage of RGC degeneration, in which RNFL birefringence precedes changes in RNFL thickness, also occurs during experimental glaucoma; initial results in 4 animals suggest that it does (Fortune et al, IOVS 2008;49:ARVO E-Abstract 3761).

The secondary purpose of this study was to determine whether abnormalities of RGC function would be associated with the intermediate stage of degeneration described above (i.e. whether altered function would also precede RNFL thinning after ONT). Three different modes of ERG testing were used to monitor retinal function in this study. The pattern of results for each mode is consistent with loss of RGC function predominantly though there was some evidence that mild disruption of function of other retinal elements may have also occurred. The photopic ff-ERG revealed a greater effect on the PhNR than on the a-wave, b-wave or OP’s. The PhNR is thought to be dependent on intact RGC function while the a-wave and b-wave represent responses of more distal retinal generators.4749 The N95 component of the PERG was affected more than the P50 component during the second week post-ONT. This pattern is again is consistent with loss of predominantly inner retinal function, particularly of RGCs.50 The mfERG HFC were affected by ONT to a greater extent than the LFC features N1, P1 and N2. This pattern is also indicative of abnormal RGC funtion.2325,51,52 It is possible that restricting the band-pass filter to isolate only the highest frequency content of these mfERG responses could provide an even more sensitive indicator and greater dynamic range over which to study effects on RGC function.52

To the extent that the photopic ff-ERG a-wave represents cone photoreceptor signaling,49,53,54 the results of this study suggest that there was a relatively mild, transient effect of ONT on function of the outer retina (though not statistically significant given the relatively small sample size and variability of the a-wave amplitude). The 10% reduction in a-wave amplitude one week after ONT, which seems to have resolved to 96% of baseline values by the second week after ONT, may have been due to the trauma associated with the orbitotomy. For the conditions of this study (background and flash intensities and chromaticities, criterion time of a-wave amplitude measurement), it is likely that the a-wave amplitude measurement reflects a relatively large contribution from hyperpolarizing second-order retinal neurons such as horizontal and off cone bipolar cells in addition to cone photoreceptor responses.53,55 Nonetheless, the results suggest that any mild outer retinal functional changes recovered almost completely by the second week after ONT.

In summary, the results of this study demonstrate that RNFL birefringence declines prior to and faster than RNFL thickness in the weeks following experimental injury of RGCs by ONT. This result suggests that disruption of the RGC cytoskeleton precedes RNFL thinning after injury. Abnormalities of RGC function were present along with decreased RNFL birefringence one week after ONT, when RNFL thickness and ONH topography were still normal. Collectively, the results are indicative of a stage of RGC dysfunction preceding changes in RNFL thickness. Further studies are underway to determine if similar intermediate stages of degeneration and abnormal function are detectable in experimental glaucoma prior to changes in RNFL thickness. Finally, although the results reported here were robust to rigorous statistical analysis and similar in all three subjects, the conclusions are based on a small sample size of n=3, which should be considered as a limitation of this study.

Acknowledgements

The authors wish to thank Roger A. Dailey, MD and Leonard A. Levin, MD, PhD for helpful consultation and assistance toward optimization of lateral orbitotomy for optic nerve transection.

Support: NIH R01-EY011610 (CFB); Glaucoma Research Foundation (BF); Legacy Good Samaritan Foundation; Heidelberg Engineering, GmbH, Heidelberg, Germany (equipment); and Carl Zeiss Meditech, Inc. (equipment).

Footnotes

Commercial relationships policy: B Fortune: F, equipment from Carl Zeiss Meditech Inc.; CF Burgoyne: F, equipment from Heidelberg Engineering, GmbH; GA Cull: None.

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